The long term goals of this project are to elucidate the mechanism of transcriptional control by heterochromatin, a structure that spans and inactivates large regions of eukaryotic genomes. Heterochromatic domains are constitutively condensed, inaccessible structures that contain hypoacetylated histones and specialized non-histone components. Many genes involved in early stages of development are permanently shut off by formation of heterochromatin or related structures. Inappropriate expression of these genes leads to abnormal development, leukemias, and other disease states. Recently, heterochromatin components have been linked to breast cancer and the control of medically relevant euchromatic genes. Inactivation of genes at the silent mating-type loci and telomeres of yeast Saccharomyces cerevisiae involves a chromatin structure, termed silent chromatin or yeast heterochromatin, that bears functional and structural similarities to the heterochromatin of higher eukaryotes. At the silent mating loci, repression requires cis-acting regulatory sequences, termed silencers, and a set of non-histone chromatin components known as the Sir proteins. A critical event during S-G2/M progression establishes a silent state that is maintained during the remainder of the cell cycle and inherited by progeny cells. To further understand the fundamental properties of heterochromatin-mediated repression, we will explore the molecular events leading to establishment of the silent state in yeast. Special focus will be placed on characterizing the changes in chromatin during establishment, and identifying the cell cycle-dependent requirement. Silent chromatin fragments will be purified and the stochiometry of silencing factors will be determined. Electron microscopy will be used to visualize higher order structural features of silent chromatin rings in vitro and real-time fluorescence microscopy will be used to identify parameters that localize silent chromatin in vivo. Genetic methods will be used to investigate how one nuclear periphery protein, Esc 1, contributes to silencing. ? ?
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